Vibrio cholerae is a facultative human pathogen that causes the severe diarrheal disease cholera. The ability of V. cholerae to cause disease is dependent upon the regulated expression of virulence factors. For example, the virulence genes encoding for production of cholera toxin and the toxin coregulated pilus appear to be induced early in infection and repressed late in infection before exiting the human gastrointestinal tract. Other genes that are induced late during infection are believed to be important for the establishment of the hyperinfective phenotype exhibited by human shed vibrios, and for survival in the aquatic ecosystem. The mechanisms that control gene expression in vivo are largely unknown. Quorum sensing has been proposed as one potential mechanism to govern late gene expression in vivo. However, the observation that many epidemic V. cholerae strains are quorum sensing insensitive suggests that other regulatory mechanisms must exist. Our laboratory has shown that diketopiperazines inhibit virulence factor production in V. cholerae. In this grant we will test the hypothesis that cyclo(Phe-Pro), a natural diketopiperazine produced by V. cholerae, functions as a signal to modulate virulence gene expression independent of the known quorum sensing systems. Two specific aims are proposed. In specific aim 1 we will characterize cyclo(Phe-Pro) and define genes that are involved in its synthesis . In specific aim 2 we will define the mechanism by which cyclo(Phe-Pro) regulates virulence factor production. These studies will shed light on a novel regulatory system that controls virulence factor production and may facilitate the future development of novel therapeutic approaches for treatment of cholera.